CN112899277B - Cotton pollen fertility-related long-chain non-coding RNA and application of target gene thereof - Google Patents

Abstract

The invention discloses a cotton pollen fertility-related long-chain non-coding RNA and application of a target gene thereof. The lncRNA and the protein provided by the invention are derived from upland cotton and consist of nucleotides shown in a sequence 1 in a sequence table and amino acids shown in a sequence 5 in the sequence table. Experiments prove that when the recombinant vectors pCLCrVA-TCONS _00473367 and pCLCrVA-GhYP 724B containing the DNA molecules shown from 6 th to 502 th in the sequence 1 of the sequence table and the DNA molecules shown from 884 th to 1379 th in the sequence 3 of the sequence table are injected into cotton cotyledons, the vegetative growth of cotton plants is not influenced, but anthers in a full-bloom stage are shrunken, the number of pollen grains is reduced, and the fertility is reduced. The invention provides a new lncRNA/gene and a method for creating a novel sterile line material, and has important significance in the aspects of creating plant hybrid seeds and utilizing heterosis.

Description

Cotton pollen fertility-related long-chain non-coding RNA and application of target gene thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of a cotton pollen fertility-related long-chain non-coding RNA TCONS _00473367 and a target gene GhYP 724B thereof.

Background

The cotton heterosis is obvious, and the cotton yield can be obviously improved by planting the hybrid cotton. The hybrid seed created by the male sterile system is time-saving, labor-saving and low in cost. Therefore, the development mechanism of the cotton male sterility is beneficial to constructing a male sterility system and is beneficial to hybrid seed selection and heterosis utilization.

Long-chain non-coding RNA (LncRNA) is a type of non-coding RNA with a regulating function, and has been proved to be involved in regulating and controlling the fertility of male organ pollen in various crops. The rice LDMAR is lncRNA with the length of 1236 bp: under long-day conditions, LDMARs of sufficient abundance are essential for normal pollen development. The increase of the methylation level of the LDMAR promoter region in the mutant plant leads to the reduction of LDMAR expression abundance, which causes the degradation of anther tapetum and causes the male sterility of rice. Maize Zm401 has a small open reading coding frame, belongs to a long non-coding RNA, and is specifically highly expressed in tapetum cells and pollen grain microspores. Silencing Zm401 causes the expression pattern of corn functional genes ZmMADS2, ZmM3-3 and ZmC5 to change, causes tapetum cell and microspore dysplasia, and influences stamen growth and anther development. To date, relatively few lncrnas have been reported in cotton, and none have been implicated in pollen fertility.

TCONS _00473367 is specifically and highly expressed in a cotton fertile material bud, a target gene GhYP 724B codes hydroxylating enzyme and regulates brassinolide synthesis, but no report that the lncRNA and protein coding gene regulates pollen fertility exists in cotton.

Disclosure of Invention

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

the invention aims to provide a cotton pollen fertility-related long-chain non-coding RNA and application of a target gene thereof.

The pollen fertility-related long-chain non-coding RNA and the target gene thereof provided by the invention are derived from upland cotton (Gossypium hirsutum L.), the name of the long-chain non-coding RNA (lncRNA) is TCONS _00473367, and the name of the target gene is GhCry 724B;

the TCONS _00473367 is the nucleotide sequence of a) or b) as follows:

a) a long-chain non-coding RNA consisting of a nucleotide sequence shown in a sequence 1 in a sequence table;

b) and (b) long-chain non-coding RNA which is derived from (a) and related to plant pollen fertility and is obtained by substituting and/or deleting and/or adding one or more nucleotide bases in the nucleotide sequence shown in the sequence 1 of the sequence table.

The protein encoded by the GhYP 724B is the protein sequence of c) or d):

c) a protein consisting of an amino acid sequence shown in a sequence 5 in a sequence table;

d) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 5 of the sequence table, is related to plant pollen fertility and is derived from the protein in the step (c);

the amino acid sequence shown in sequence 5 of the sequence table consists of 493 amino acid residues.

In order to facilitate the purification of the protein in c), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 5 of the sequence listing is linked with a tag as shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue(s) of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in d) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of d) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown by the nucleotide sequence from position 1 to position 1482 of the sequence 3 in the sequence table, and/or by carrying out missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or the 3' end thereof.

The coding gene of the protein also belongs to the protection scope of the invention.

The coding gene of the protein is any one of the following DNA molecules 1) to 6):

1) DNA molecules shown from 1 st to 1482 nd in a sequence 3 of a sequence table;

2) DNA molecules shown from 1 st to 3334 th in a sequence 4 of a sequence table;

3) DNA molecules shown in sequence 3 of a sequence table;

4) DNA molecules shown in sequence 4 of a sequence table;

5) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in 1) or 2) or 3) or 4) and encoding the protein of claim 1;

6) a DNA molecule which hybridizes under stringent conditions with a DNA sequence defined in 1) or 2) or 3) or 4) or 5) and which encodes a protein according to claim 3.

Sequence 2 of the sequence table consists of 1101 deoxynucleotides, is a full-length sequence of Gossypium hirsutum L long non-coding RNA TCONS _00473367, and comprises two exons (1-480 th position and 780-1101 th position) and an intron.

The sequence 4 of the sequence table is cotton genome DNA corresponding to the GhYP 724B gene, the total length is 3334bp, and the cotton genome DNA consists of 9 exons and 8 introns, wherein the 9 exons are respectively: the 1-224 bit, the 366-691 bit, the 807-960 bit, the 1159-1438 bit, the 1537-1627 bit, the 1841-1920 bit, the 2085-2192 bit, the 3002-3124 bit and the 3231-3334 bit.

The sequence 1 in the sequence table is identified as long-chain non-coding RNA, and the identification strict conditions are as follows: the length of the transcript in the sequence 1 of the sequence table is 802bp, which exceeds 200bp defined by the lncRNA standard;

first, with the CPAT website:http://lilab.research.bcm.edu/cpat/predicting the coding ability score of sequence 1 in the sequence table, wherein the result shows that score is-0.0681, and the sequence is non-coding RNA;

then, through the CNCI website:https://en.bioinfo.org/software/cncipredicting the coding ability score of the sequence 1 in the sequence table, wherein the result shows that score is-0.0328, and the sequence is non-coding RNA;

again, using the CPC website:http://cpc.cbi.pku.edu.cn/predicting the coding ability score of the sequence 1 in the sequence table, wherein the result shows that score is-1.2201 and the sequence is non-coding RNA;

the result of Pfam (http:// Pfam. janelia. org /) analysis shows that the sequence 1 transcript in the sequence table can not be aligned to the HMM statistical model of the amino acid sequence, and further proves that the transcript is potential lncRNA.

The expression level (FPKM) of the transcript in the bud of fertile material 2074B has the values of 5.5708, 7.2964 and 5.6904 respectively; the results are combined to fully prove that the transcript of the sequence 1 in the sequence table is lncRNA.

Sequence 3 of the sequence table is positioned at the downstream 28kb of sequence 1 of the sequence table and is regulated and controlled by sequence 1 of the sequence table.

The recombinant vector, the expression cassette, the transgenic cell line, the recombinant bacterium or the recombinant virus vector containing the long-chain non-coding RNA (lncRNA) and the target gene thereof also belongs to the protection scope of the invention.

The existing plant expression vector can be used for constructing a recombinant expression vector containing the long non-coding RNA (lncRNA) and a target gene thereof.

The plant expression vector comprises virus-mediated gene silencing vectors, such as pCLCrV, TbCSV, TMV, PVX, TRV, TGMV, CbLCV, ACMV, STMV, TYLCCNV and the like.

The plant expression vector can comprise conserved sequences of exon regions of the exogenous genes, and the length of the fragment is set to be between 150bp and 510 bp.

In addition, when the gene is used for constructing a plant expression vector, a CHLI (cotton magnesium ion chelating enzyme subunit I) gene silencing vector is constructed at the same time and is used as a positive control of the next experiment, so that the gene silencing efficiency is convenient to detect; at the same time, the empty vector pCLCrVA for gene silencing mediated by the transformed virus is used as a negative control.

The recombinant vector can be a recombinant expression vector obtained by inserting the lncRNA or a target gene thereof into a vector pCLCrVA, and the length of a target fragment is set to be between 495bp and 505 bp. Specifically, the DNA molecules shown from the 6 th site to the 502 th site in the sequence 1 of the sequence table are inserted into a vector pCLCrVA to obtain a recombinant expression vector; inserting the DNA sequence from 884 th site to 1379 th site in the sequence 3 of the sequence table into a vector pCLCrVA to obtain a recombinant expression vector.

The recombinant vector is prepared according to the following method: and (3) carrying out double enzyme digestion on the vector pMD18-T containing the lncRNA or the gene by using Spe I and Pac I, recovering a target fragment with the size of 0.5kb, and connecting the target fragment with the framework of the vector pCLCrVA subjected to double enzyme digestion by using Spe I and Pac I to obtain the recombinant vector.

The invention protects the application of the long non-coding RNA (lncRNA) and the target gene thereof in the plant body to regulate and control the pollen fertility of the plant.

Another objective of the invention is to provide a method for breeding transgenic plants, which is to knock out the long non-coding RNA (lncRNA) or the target gene thereof in the target plants to obtain transgenic plants of male sterile plants.

The invention also provides a method for creating cotton hybrid, which comprises the process of knocking out the created male sterile female parent of the long non-coding RNA (lncRNA) and the target gene thereof.

In both of the above methods, the knockout is effected by the recombinant vector.

In both methods, the recombinant vector is introduced into the plant by the cotyledon injection method.

In the above applications and methods, the plant of interest is a fertile cotton material.

The invention has the beneficial effects that:

experiments prove that when the recombinant vector pCLCrVA-TCONS _00473367 containing the DNA molecules shown from the 6 th site to the 502 th site in the sequence 1 of the sequence table is injected into cotton cotyledons, cotton plants grow normally in nutrition and pollen fertility is reduced in the full-bloom stage. Detecting the expression pattern of a target sequence, and finding that TCONS _00473367 and a target gene GhYP 724B in the VIGS plant are obviously reduced; the recombinant vector pCLCrVA-GhYP 724B containing the DNA molecules shown from the 884 th site to the 1379 th site in the sequence 3 of the sequence table is injected into cotton cotyledons, so that the pollen fertility of cotton plants is also reduced, and the male sterility phenomenon is shown. The invention provides an lncRNA and a targeted protein coding gene thereof for creating a new cotton male sterile line, and has important significance for further creating cotton hybrid through a male sterile system.

Drawings

The invention has the following drawings:

FIG. 1 shows the expression pattern of TCONS _00473367 and its target gene GhYP 724B in different materials and different tissue sites:

a is a diagram of a semi-quantitative PCR electrophoresis diagram and a fluorescence quantitative bar diagram of specific expression of TCONS _00473367 in fertile material tissues;

b is a diagram of the expression pattern of TCONS _00473367 in different materials and different tissues;

and the c picture shows the expression pattern of the GhYP 724B in different materials and different tissues.

FIG. 2 is the structural schematic diagram of the recombinant plasmid pCLCrVA-TCONS _00473367 and the recombinant plasmid pCLCrVA-GhCry 724B.

FIG. 3 is the structural diagram of the helper plasmid pCLCrVB of the virus-mediated gene silencing vector.

The target fragment described in FIG. 2 was inserted at both the Spe I and Pac I multiple cloning sites. The VIGS vector consists of two parts, wherein AL1-AL4 in pCLCrVA respectively encode coat protein, replication associated protein, transcription activating protein and replication enhancing protein; BV1 and BC1 in pCLCrVB function as a nuclear shuttle protein and a motor protein, respectively.

FIG. 4 is a schematic diagram showing the expression pattern of TCONS _00473367 in a recipient plant 2074B, a transgenic empty vector pCLCrVA plant, a transgenic pCLCrVA-TCONS _00473367 plant and a transgenic pCLCrVA-GhYP 724B plant, wherein a column diagram is a qRT-PCR detection result, and a gel electrophoresis diagram is a semi-quantitative PCR detection result;

FIG. 5 shows the expression pattern of GhCRYP 724B in recipient plant 2074B, empty vector pCLCrVA, pCLCrVA-TCONS _00473367 and pCLCrVA-GhCRYP 724B, the column diagram is qRT-PCR detection result, and the gel electrophoresis diagram is semi-quantitative PCR detection result.

FIG. 6 is an observation of the phenotype of the floral organs of transgenic cotton:

a picture is a picture of flowers of a transgenic empty vector pCLCrVA cotton plant in the full-bloom stage;

b is a picture of the flower of the cotton plant at the full-bloom stage of the transgenic pCLCrVA-TCONS _ 00473367;

c is the flower map of the full bloom stage of the transgenic cotton plant pCLCrVA-GhYP 724B;

d is the anther map of the empty vector pCLCrVA cotton plant in the full-bloom stage;

the e picture is the anther picture of the full-bloom stage of a transgenic pCLCrVA-TCONS _00473367 cotton plant;

f is the anther map of the cotton plant at the full-bloom stage of the transgenic pCLCrVA-GhYP 724B;

g picture is transferred empty carrier pCLCrVA cotton plant full-bloom stage flowerPowder I ₂ -KI staining pattern;

h picture is pCLCrVA-TCONS _00473367 cotton plant full-bloom pollen I ₂ -KI staining pattern;

i picture is the pollen I of cotton plant at full bloom stage transformed with pCLCrVA-GhYP 724B ₂ -KI staining pattern.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1, TCONS-00473367 and its target Gene GhCryP 724B expression analysis

Step 11 different tissue harvesting and preservation of different cotton materials

Fertile material 2074B (Su Cotton 20; Liu et al, Over-expression of transcription factor GhWRI1 in upland cotton. biologica Plantarum 2018.62: 335) 342 publicly available from Chinese university of agriculture) Leaf (Leaf), flower bud from archesporial cell stage to pollen mother cell stage (FB-I), flower bud from meiotic stage to binuclear stage (FB-II), flower bud at pollen grain maturity (FB-III), one day bud before flowering (Bud), full bloom Petal (Petal), full bloom Stigma (Stigma), full bloom Anther (Anther), and 10 day after flowering Seed (Seed) are first taken for tissue specific expression analysis of TCONS _ 00473367. In addition, the buds of sterile line 2074A from meiotic stage to binuclear stage, the buds of fertile material E5903 from meiotic stage to binuclear stage, and F are selected ₁ Bud from meiosis stage to binuclear stage of-A (2074A × E5903) for specific and tissue-specific expression analysis of TCONS _00473367 and GhYP 724B cotton species. All the samples were snap frozen in liquid nitrogen and stored at-80 ℃ for RNA extraction.

Step 12 extraction of Total RNA and cDNA obtaining

The feature and mechanism of abortion of cytoplasmic male sterile line 2074A of cotton are studied by using improved CTAB-ammonium acetate method (Nie Hushushuai, Nie Hushuai, Japan thesis of doctor's university: [ doctor academic thesis]2019) of the step 11 of storing the material were separately extracted from the total RNAUsing PrimeScript ^TM The cDNA was obtained by reverse transcription using RT reverse transcription kit (TaKaRa, Dalian, China).

Step 13 Semi-quantitative PCR (Semi-quantitative RT-PCR) and Real-time fluorescent quantitative PCR (Real-time PCR)

Diluting cDNA obtained by reverse transcription in the step 12 by 6 times and then using the diluted cDNA as a template of semi-quantitative PCR and real-time fluorescent quantitative PCR. Designing specific primers TC67-F and TC67-R according to the sequence of the TCONS _00473367 transcript; designing specific primers CYP724-F and CYP724-R according to a conserved sequence of GhYP 724B; GhUBQ7 was used as an internal reference gene.

Real-time fluorescent quantitative PCR was performed on an ABI 7500 real-time fluorescent quantitative PCR instrument, and 3 replicates were set for one parallel experiment. The method reported by Livak KJ and Schmittgen TD (2001), 2 ^-ΔΔCT And calculating the relative expression amount. The results are shown in FIG. 1.

ΔΔC _T ＝(C _T.Target -C _T.UBQ7 ) _{Time x} -(C _T.Target -C _T.UBQ7 ) _{Time 0}

Time x denotes an arbitrary Time point, Time ₀ Represents the expression of the target gene in an amount of 1 time the amount was corrected by UBQ 7.

The sequences of the primers TC67-F/TC67-R and CYP724-F/CYP724-R are as follows:

TC 67-F: 5'-AGCAACGCCTTCCCTTTCA-3' (corresponding to the 30 th to 48 th position of the exon region in the sequence 1 in the sequence table);

TC 67-R: 5'-TCAAGATACCGTCGTGAGCAA-3' (corresponding to position 185-205 of the exon region in sequence 1 of the sequence listing).

CYP 724-F: 5'-TCAAGGGTAAGGGTGGGACT-3' (corresponding to position 97-116 of exon region 3 in the sequence table);

CYP 724-R: 5'-GGGGAGAAGAACAAATGGG-3' (corresponding to position 245-262 of the exon region of sequence 3 in the sequence listing).

The primer sequences of the internal reference gene GhUBQ7 are as follows:

GhUBQ7-F：5’-GAAGGCATTCCACCTGACCAAC-3’

GhUBQ7-R：5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’

the results of a chart in fig. 1 show that TCONS _00473367 is specifically highly expressed in the bud at the sporocyte stage and the meiosis stage to the binuclear stage, and is expressed in lower abundance in other tissues and organs; the results of the b-diagram in FIG. 1 and the c-diagram in FIG. 1 show that TCONS _00473367 and GhYP 724B are expressed in higher amount in the fertile material buds, and are expressed in lower abundance in the sterile material buds and other tissues of the fertile material.

Example 2 cloning of Long-chain non-coding RNATCONS _00473367 and protein coding Gene GhCryP 724B fragment of Cotton and construction of VIGS vector

Using bud from meiosis stage to binuclear stage of cultivated species of land cotton (Gossypium hirsutum L.) and of Sucus Gossypium 20(2074B) as material to extract total RNA, reverse transcribing to obtain cDNA, and performing PCR amplification by using the cDNA as template and the primers of VIGS67-F/VIGS67-R and VIGS724-F/VIGS 724-R.

The primer sequences for the PCR amplification are as follows:

primer VIGS 67-F: 5'-CCTTAGGAGAAGAGGATGATTCGCT-3';

primer VIGS 67-R: 5'-TTCATCATGAAATCACAGTGAACCT-3' are provided.

Primer VIGS 724-F: 5'-TGGATTCTTTATTGGGTGGC-3', respectively;

primer VIGS 724-R: 5'-CCTGAAGTTTTGAACAAGGTGG-3' are provided.

The PCR primer sequence is characterized in that a Spe I (ACTAGT) enzyme cutting site sequence is added at the 5 'end of an upstream primer, and a Pac I (TTAATTAA) enzyme cutting site sequence is added at the 5' end of a downstream primer.

Reaction system for PCR amplification (25. mu.l): mu.l cDNA, 1. mu.l upstream and downstream primer mix (10. mu.M), 2.5. mu.l 10 × LA buffer, 2. mu.l dNTP (2.5mM each), 0.3. mu.l LA-Taq enzyme, 17.2. mu.l ddH ₂ O。

Reaction procedure for the above PCR amplification: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30S, annealing at 60 ℃ for 30S, and extension at 72 ℃ for 30S, and 32 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

And respectively carrying out 1% agarose gel electrophoresis detection on the PCR amplification products, respectively recovering target fragments of about 500bp, connecting the target fragments to a pMD18-T (TAKARA, D101A) cloning vector, transforming the connecting products into an Escherichia coli DH5 alpha strain after 12h, screening positive clones by adopting a blue-white screening method, and sending the positive clones to a company for sequencing after PCR detection.

The sequencing result is compared with a target sequence of a reference genome, and the result shows that the positive cloning vector is named pMD18-T-TCONS _00473367, and a DNA fragment with the length of 497bp, which is shown by the 6 th to 502 th nucleotide sequences of a sequence 1 in a sequence table, is inserted into the vector pMD 18-T; the positive cloning vector is named as pMD 18-T-GhCry 724B, and is a DNA fragment with the length of 496bp, which is shown by the nucleotide sequence from the 884 th site to the 1379 th site of the sequence 3 in the sequence table, is inserted into the vector pMD 18-T.

Amplifying and shaking the monoclonal bacteria liquid with correct sequencing, respectively extracting plasmid DNA, then, carrying out enzyme digestion on the plasmid DNA by using restriction enzymes Spe I and Pac I, and recovering an enzyme digestion product; simultaneously, the virus-mediated gene silencing vector pCLCrVA is cut by restriction enzymes Spe I and Pac I, and a vector framework is recovered; connecting the enzyme digestion product of the target fragment in the step with the enzyme digestion product of the carrier skeleton; after overnight at 16 ℃, the ligation product is transformed into an escherichia coli DH5 alpha strain, overnight culture is carried out at 37 ℃, and a single clone is selected for PCR detection; positive cloning recombinant plasmids pCLCrVA-TCONS _00473367 and pCLCrVA-GhYP 724B are respectively extracted for double enzyme digestion verification. The result proves that the recombinant plasmid is a DNA fragment with the length of 497bp, which is shown from the 6 th position to the 502 th position of the sequence 1 in a sequence table, inserted between the Spe I enzyme cutting sites and the Pac I enzyme cutting sites of the vector pCLCrVA, and a DNA fragment with the length of 496bp, which is shown from the 884 th position to the 1379 th position of the sequence 3 in the sequence table, inserted between the Spe I enzyme cutting sites and the Pac I enzyme cutting sites of the vector pCLCrVA.

The pCLCrVA vector backbone described above was modified and provided by the subject group of the teacher from Zhang Tong of the plant science institute of Chinese university (Gu et al. A versatil system for functional analysis of genes and microRNAs in cotton. plant Biotechnol Journal,2014,12(5): 638-.

Example 3 Virus-mediated Gene silencing of Cotton TCONS-00473367 and GhYP 724B

Step 31 construction of recombinant Agrobacterium

Taking the recombinant plant expression vectors pCLCrVA-TCONS _00473367 and pCLCrVA-GhCRYP 724B prepared in example 2, as well as a positive control vector pCLCrVA-CHLI, an empty vector pCLCrVA and an auxiliary vector pCLCrVB, transforming competent cells of Agrobacterium tumefaciens GV3101 by a freeze-thaw method, wherein the competent cells are purchased from Beijing Tuolinfang technology Limited, cultured for 3-4h at 28 ℃ in a liquid YEP medium without antibiotics by shaking, and then coated on YEP solid culture containing 50 mug/ml kanamycin sulfate and 50 mug/ml rifampicin for screening culture; performing colony PCR (polymerase chain reaction) by using primers VIGS67-F/VIGS67-R and VIGS724-F/VIGS724-R in example 2 respectively, then identifying positive single clones, and respectively naming the correctly identified agrobacterium, namely the recombinant agrobacterium containing recombinant plant expression vectors pCLCrVA-TCONS _00473367 and pCLCrVA-GhCyp724B as GV3101/pCLCrVA-TCONS _00473367 and GV3101/pCLCrVA-GhCyp 724B; the positive control vector, the empty vector and the auxiliary vector are named as GV3101/pCLCrVA-CHLI, GV3101/pCLCrVA and GV 3101/pCLCrVB.

Step 32 cotyledon injection method to obtain target lncRNA and cotton plant with gene silencing

Step 321 preparation of agrobacterium infection liquid

The recombinant Agrobacterium tumefaciens GV3101/pCLCrVA-TCONS _00473367, GV 3101/pCLCrVA-GhCry 724B, GV3101/pCLCrVA-CHLI, GV3101/pCLCrVA and GV3101/pCLCrVB obtained in step 31 were inoculated into 5ml of YEP liquid medium containing 50. mu.g/ml kanamycin sulfate and 50. mu.g/ml rifampicin, shaken overnight, then expanded into 500ml of YEP liquid medium, and cultured at 28 ℃ to OD ₆₀₀ 0.8 to 1.5; centrifugally collecting thalli, and suspending the thalli by using an infection solution, wherein the infection solution comprises: MES: 10mmol/L, MgCl: 10mmol/L, acetosyringone: 200 mu mol/L; to OD ₆₀₀ In the range of 0.8 to 1.5, an Agrobacterium suspension was obtained.

Step 322 Cotton cotyledon injection

Soaking delinted Suzhou cotton 20(2074B) seeds in tap water overnight, after the seeds are exposed to white, sowing the seeds in a flowerpot with the diameter of 30cm, and culturing at 28 ℃. After the cotyledon of the cotton is flattened, agrobacterium suspension GV3101/pCLCrVA-TCONS _00473367, GV 3101/pCLCrVA-GhCRYP 724B, GV3101/pCLCrVA-CHLI and GV3101/pCLCrVA prepared in the step 321 are respectively mixed with the GV3101/pCLCrVB suspension in the same volume to obtain a mixed solution, and the mixed solution is stood for 3 hours at room temperature. The mixture was injected into the leaf from the back of the cotton cotyledon by a syringe to fill the whole cotyledon with the suspension. The cotton plants after injection are placed in a greenhouse at 28 ℃ and cultured in 16h/8h light and dark cycle.

Step 323 Gene silencing efficiency test

After injection is completed for 1-2 weeks, observing the growth condition of the true leaves of the GV3101/pCLCrVA-CHLI injected plants, if the true leaves turn yellow, indicating that CHLI gene is silenced, and the VIGS system is effective. After the bud of the transgenic cotton plant appears, the bud from the meiosis stage to the binuclear stage is taken, the bud leaves, sepals, petals and ovules are removed, the total RNA is extracted, the cDNA is synthesized by the method in reference example 1, and semi-quantitative PCR and real-time fluorescent quantitative PCR detection are carried out. The primers used were as described in example 1.

The detection results are shown in fig. 4 and fig. 5, in the cotton plant injected with GV3101/pCLCrVA-TCONS _00473367 (VIGS-TCONS _00473367), the expression pattern of TCONS _00473367 is significantly reduced compared to that of the recipient plant, i.e., pericotton 20(2074B) and the cotton plant injected with GV3101/pCLCrVA (VIGSA), and in addition, the expression pattern of the target gene ghcrypt 724B of TCONS _00473367 is also significantly reduced. In the cotton plant (VIGS-GhCRYP 724B) injected with GV 3101/pCLCrVA-GhCRYP 724B, the expression pattern of GhCRYP 724B is significantly reduced compared with that of the recipient plant, namely Suzhou cotton 20(2074B) and the GV 3101/pCLCrVA-injected cotton plant, while the expression pattern of TCONS _00473367 is not significantly changed. The results show that the GV3101/pCLCrVA-TCONS _00473367 can remarkably silence the expression abundance of TCONS _00473367 and TCONS _00473367 can positively regulate GhCRYP 724B, so that the expression pattern of the GhCRYP 724B is also remarkably reduced; GhCRYP 724B expression abundance can be remarkably silenced by injecting GV 3101/pCLCrVA-GhCRYP 724B, and GhCRYP 724B has no inverse regulation effect on TCONS _ 00473367.

Step 33 silencing TCONS _00473367 and its target Gene GhCryP 724B Cotton phenotypic characterization

The phenotype of the silenced TCONS _00473367 cotton plant is observed, and the result shows that compared with a receptor and a negative control plant, the silenced TCONS _00473367 has no influence on the vegetative growth of cotton; when the plants enter the full-bloom stage, the anthers of the receptor and the negative control plants are full, pollen grains completely shed in the pollen-scattering peak stage and pollination is completed; while the anther of the TCONS _ 00473367-silenced cotton plant is shriveled and is scatteredNo fertile pollen grains are scattered in the peak period; mashing anther, and processing pollen I ₂ And (4) KI staining, and microscopic observation shows that the silent TCONS-00473367 cotton plant has fewer pollen grains, irregular shape and lighter coloring and shows the characteristic of male sterility. Specifically silencing ghcrypt 724B, cotton plants showed the same phenotypic trait as silencing TCONS _00473367, normal vegetative growth, anther shriveling, irregular shape of pollen grains, lighter coloration and fewer numbers, and the results are shown in fig. 6, panels a-i.

The results are combined to show that when TCONS _00473367 is silenced, the expression of a target gene GhYP 724B is obviously reduced, and a plant shows a male sterility phenomenon; the GhYP 724B is specifically silenced, and male sterility also occurs in plants; the above results were confirmed in two-year repeated experiments.

To summarize: the long-chain non-coding RNA TCONS _00473367 influences the pollen fertility of cotton plants by regulating the expression mode of GhYP 724B.

Those not described in detail in this specification are within the skill of the art.

SEQUENCE LISTING

<110> university of agriculture in China

<120> application of cotton pollen fertility-related long-chain non-coding RNA and target gene thereof

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 802

<212> DNA

<213> upland cotton (Gossypium hirsutum L.)

<400> 1

aacatcctta ggagaagagg atgattcgct agcaacgcct tccctttcat ggtagcacaa 60

acatcaaagt gccacttttt tttaaatcgg agctaaaagg gcaaaacatg ccaatagatt 120

cagggatgct acgcaacatg ctaagcctga tcccacataa atcaagacca tgggcgaatt 180

gtatattgct cacgacggta tcttgaatgt taacattttt agaaaaaagt ttgttccgcc 240

tctgcataag cacgatcgac agttgcctac tactacctca acatcatatt ttgcttgaaa 300

ccttcttaca accaacttat tttgcttttg actgagtctc aagacaatgg ccctattgca 360

tgtacgcaaa agcattgcca ttcctctctt actccgacaa caatcgctct ttccaaagat 420

cgacttccta ctcacatgat tttacccgac ggtgttcttc ttcacatgaa tttgaagtag 480

gttcactgtg atttcatgat gaaaacaaaa atgctggtaa gtggcggtca caaaacagaa 540

atatatgaat aaatatttgg tagataataa gggagttgag attttatttc ttttttttca 600

tcaattaata tattaaataa ttattctatg taaggttaat taaggtaaat taatttattt 660

ggtatatata atatttttat ttatttgtat tattatctca aatttttatg tcatgttatt 720

tttaggttta atccaccaat tgatttctaa actatctttt tttttctaac ttacttcata 780

ttttttggtc acaagtgcta cc 802

<210> 2

<211> 1101

<212> DNA

<213> upland cotton (Gossypium hirsutum L.)

<400> 2

aacatcctta ggagaagagg atgattcgct agcaacgcct tccctttcat ggtagcacaa 60

acatcaaagt gccacttttt tttaaatcgg agctaaaagg gcaaaacatg ccaatagatt 120

cagggatgct acgcaacatg ctaagcctga tcccacataa atcaagacca tgggcgaatt 180

gtatattgct cacgacggta tcttgaatgt taacattttt agaaaaaagt ttgttccgcc 240

tctgcataag cacgatcgac agttgcctac tactacctca acatcatatt ttgcttgaaa 300

ccttcttaca accaacttat tttgcttttg actgagtctc aagacaatgg ccctattgca 360

tgtacgcaaa agcattgcca ttcctctctt actccgacaa caatcgctct ttccaaagat 420

cgacttccta ctcacatgat tttacccgac ggtgttcttc ttcacatgaa tttgaagtag 480

gtatttctaa gttctgtttc gatataatta agggtttata tgcataaaat aaatcttgga 540

tcccatatca acttaatttg tataatttgt tgatacatca cattcgttga ggcgaagggg 600

ttttatttta taaattatta gattatttag aagaaaaaaa acatgtgttt cacttgggaa 660

aaagtcggtt caatcaccac ataacaacaa aaacaagact tcactattcc atgcatggta 720

ataactcgtt gaattaaaga atattaatga tggtaaacta attactttta tgattacagg 780

ttcactgtga tttcatgatg aaaacaaaaa tgctggtaag tggcggtcac aaaacagaaa 840

tatatgaata aatatttggt agataataag ggagttgaga ttttatttct tttttttcat 900

caattaatat attaaataat tattctatgt aaggttaatt aaggtaaatt aatttatttg 960

gtatatataa tatttttatt tatttgtatt attatctcaa atttttatgt catgttattt 1020

ttaggtttaa tccaccaatt gatttctaaa ctatcttttt ttttctaact tacttcatat 1080

tttttggtca caagtgctac c 1101

<210> 3

<211> 1482

<212> DNA

<213> upland cotton (Gossypium hirsutum L.)

<400> 3

atggctgaag gtgattcttg ctgggtggtt gttttggttg ggggagttgt tggttttatt 60

ctagttgttg tgttgaacca tttctggcct ttgctcttca agggtaaggg tgggactgtc 120

cccaagggga gttttggatg gcctttactt ggtgaaacat ttagcttctt aaagcctcac 180

tcttccaatt ctgtgggggc gtttcttcat gatcattgtt ctaggtatgg gaaggtgttc 240

aaatcccatt tgttcttctc ccccacagtg gtatcatgtg acccagagct aaactatttc 300

atacttcaaa ctgaaggcaa gctgttcgag tgtagttatc caaagcctat ccatggcatc 360

ttgggcaagg tttcaatgct tgtggcagtg ggagacactc acaagaggct cagaaatgca 420

gcactctcac tggtcaccat taccaaatca aagcctgagt ttctccatga cattgaaaac 480

atagccattc aaattctgga ctcatggaaa aataaaccac aagtcatctt ctgtgaagag 540

gctagaaagt ttacattcaa tgtaatagta aagcaagtgt tagggttgac accacaagag 600

ccagagactt cagaaattct agaagatttt ctcactttta tgagaggcct catctctctc 660

cctctctata ttcctggaac cccatatgca agagctgttc aggctagaag cagaatatct 720

tctactgtca aagctattgt agagaaaaga agagcaggaa gaagaaataa taataataat 780

gatgatgatg atgataataa taattctaaa aaaaacagtg atttcggagg aatccttcta 840

tctgttgata ccttatctga agatgaaaaa gtgagctttg ttttggattc tttattgggt 900

ggctatgaaa ctacttccct tttgatgtgt atggtgattc atttcttaag ccactcccca 960

gctgcattgc aacagttaaa gcaagaacat cttaaaataa ggagcatgaa gcagaaacat 1020

gatgatcatt tggactggga ggattataag aaaatggaat tcactcaata tgtcatcaat 1080

gaagctctca gatatggcaa cgttgtcaaa ttcgttcacc gaaaggccct taaagatgtc 1140

aaatataaag gttacctaat tccatcagga tggaaggtcc tacctgtttt cactgcagtt 1200

catttagacc catctctcca tgcaaatgct acccagttcc atccatggcg gtgggagagc 1260

caggatccca catgcaaaaa atttacaccc tttggaggtg ggtcaagatg ctgtcctgga 1320

tctgacctag ccaaggttga ggttgctttc ttcctccacc accttgttca aaacttcagg 1380

tggaaaacag aaggtgagga tcaacctata gcatacccct atgtcgaatt tcaaagagga 1440

ttggttctga atgtggatcc atgttcggaa acaactatgt ag 1482

<210> 4

<211> 3334

<212> DNA

<213> upland cotton (Gossypium hirsutum L.)

<400> 4

atggctgaag gtgattcttg ctgggtggtt gttttggttg ggggagttgt tggttttatt 60

ctagttgttg tgttgaacca tttctggcct ttgctcttca agggtaaggg tgggactgtc 120

cccaagggga gttttggatg gcctttactt ggtgaaacat ttagcttctt aaagcctcac 180

tcttccaatt ctgtgggggc gtttcttcat gatcattgtt ctaggtaagt ttataactta 240

actcacatca catgtatata tatgcctttt gtatgatgat ttttcactcc ttcagctgtg 300

atgtatatat atatatgtgt gtgtgcgtgg agtaatctaa aatgaaaatt ggtattgaaa 360

tttcaggtat gggaaggtgt tcaaatccca tttgttcttc tcccccacag tggtatcatg 420

tgacccagag ctaaactatt tcatacttca aactgaaggc aagctgttcg agtgtagtta 480

tccaaagcct atccatggca tcttgggcaa ggtttcaatg cttgtggtag tgggagacac 540

tcacaagagg ctcagaaatg cagcactctc actggtcacc attaccaaat caaagcctga 600

gtttctccat gacattgaaa acatagccat tcaaattctg gactcatgga aaaataaacc 660

acaagtcatc ttctgtgaag aggctagaaa ggtatagtac cactagtatt aataaataat 720

aatgttaaag aatctaatca aattctaatg gaatgtttct tacttttttt attttttcct 780

tttgtcaact tttggttcca tgtatagttt acattcaatg taatagtaaa gcaagtgtta 840

gggttgacac cacaagagcc agagacttca gaaattctag aagattttct cacttttatg 900

agaggcctca tctctctccc tctctatatt cctggaaccc catatgcaag agctgttcag 960

gtactcaaca aaagcactgt attgatagct ttgtttatta aattaaatga aacaataata 1020

attatggtgt ggtttaataa atggtttatg agagacttag cttagctcta tatatctctc 1080

tccttttttt ctttttattg tttttggctg caaaaaggaa gcatgtgatc atcaacttta 1140

cctttgacta tctttacagg ctagaagcag aatatcttct actgtcaaag ctattgtaga 1200

gaaaagaaga gcaggaagaa gaaataataa taataatgat gatgatgatg ataataataa 1260

ttctaaaaaa aacagtgatt tcctagaaat ccttctatct gttgatacct tatctgaaga 1320

tgaaaaagtg agctttgttt tggattcttt attgggtggc tatgaaacta cttccctttt 1380

gatgtgtatg gtgattcatt tcttaagcca ctccccagct gcattgcaac agttaaaggt 1440

aaccccaccc ttgtattcta atttccatca atttcatgca gatcttctat tattaatctc 1500

taaatctttt taattatgtt ttgaactaaa aatacagcaa gaacatctta aaataaggag 1560

catgaagcag aaacatgatg atcatttgga ctgggaggat tataagaaaa tggaattcac 1620

tcaatatgta agaaattttc tatatatata tatgttcttt ttcatacttt aatatataaa 1680

aaatagaaca caactttttc ttctaatacc ccaacaaaga agaaaaaaat acaactttgt 1740

tctgatacac ctttttctag catgtttgga ataatgatgt gattttataa ccttttattt 1800

tttctctaat gcatgcttgc ttttcttttt catgcttgta ggtcatcaat gaagctctca 1860

gatatggcaa cgttgtcaaa ttcgttcacc gaaaggccct taaagatgtc aaatataaag 1920

gttttgttat ttatttattt cgatataaaa atgttattag ttctaaaata acaatgtgca 1980

atatgattcg tgattcaata taataatgca gtgcatgaac ttatagatat tgtagtttat 2040

acttctatta taagatatat aacccaaagt tttgggttga aacaggttac ctaattccat 2100

caggatggaa ggtcctacct gttttcactg cagttcattt agacccatct ctccatgcaa 2160

atgctaccca gttccatcca tggcggtggg aggtaaatta ctcatctcat gccctaaatg 2220

gggtacccat ggcctcatct aagtaatggc tgaaagggac catgtcccta actttcactc 2280

aaaacttatc ctattttttt aatttcaact ttcatatgtt catattgggc tcctccaaaa 2340

tctgaagtca aaatataaaa gtgatcactt ataatcattc ccctatagaa aaataatact 2400

aataataatg atgattatag tatgttttaa agtaaaagtt tctttttatt tttacaattt 2460

ctaattttta agttgttgaa atgaagttct ggttctgaaa ctgggggcca tccctcggaa 2520

attttgatct gtttgtagca ttattttgtt gaaattgtgg ggggtggggg accttataaa 2580

ttgtaggaca aagtgacgac ttttcatcag aaatttggag atacgcacca ttttttttgt 2640

tagattcatg aaaatagcct ttcactgtac cttatggcca tgacagatat ttggtacacc 2700

aaacctaaaa taccttacaa tggatccgat tgaccaactt cttgatttac acattcatta 2760

ccacctaaaa caaaataaag aaacatgttt tgttgcattt cagaaacccg tgttgatttt 2820

tcaaaacttc caactatcaa atttcttgaa gtagaaatgc tttgttaatg aaagatgatc 2880

tttcaaaccc gagctcaact gggttcttgt aatcatgtga catttagtaa gttcgagttc 2940

aagtccacct ggactctttt gtggggaaaa cgtacaagat tatttccatg tttttgatgc 3000

agagccagga tcccacatgc aaaaaattta caccctttgg aggtgggtca agatgctgtc 3060

ctggatctga cctagccaag gttgaggttg ctttcttcct ccaccacctt gttcaaaact 3120

tcaggtgatt ttcttatatg tatatccagt aaacaaaatc agcactagtg ctattttctt 3180

cttcaacttt aaaaatccca atttactgta acatagtgac aaattttgta ggtggaaaac 3240

agaaggtgag gatcaaccta tagcataccc ctatgtcgaa tttcaaagag gattggttct 3300

gaatgtggat ccatgttcgg aaacaactat gtag 3334

<210> 5

<211> 493

<212> PRT

<213> upland cotton (Gossypium hirsutum L.)

<400> 5

Met Ala Glu Gly Asp Ser Cys Trp Val Val Val Leu Val Gly Gly Val

1 5 10 15

Val Gly Phe Ile Leu Val Val Val Leu Asn His Phe Trp Pro Leu Leu

20 25 30

Phe Lys Gly Lys Gly Gly Thr Val Pro Lys Gly Ser Phe Gly Trp Pro

35 40 45

Leu Leu Gly Glu Thr Phe Ser Phe Leu Lys Pro His Ser Ser Asn Ser

50 55 60

Val Gly Ala Phe Leu His Asp His Cys Ser Arg Tyr Gly Lys Val Phe

65 70 75 80

Lys Ser His Leu Phe Phe Ser Pro Thr Val Val Ser Cys Asp Pro Glu

85 90 95

Leu Asn Tyr Phe Ile Leu Gln Thr Glu Gly Lys Leu Phe Glu Cys Ser

100 105 110

Tyr Pro Lys Pro Ile His Gly Ile Leu Gly Lys Val Ser Met Leu Val

115 120 125

Ala Val Gly Asp Thr His Lys Arg Leu Arg Asn Ala Ala Leu Ser Leu

130 135 140

Val Thr Ile Thr Lys Ser Lys Pro Glu Phe Leu His Asp Ile Glu Asn

145 150 155 160

Ile Ala Ile Gln Ile Leu Asp Ser Trp Lys Asn Lys Pro Gln Val Ile

165 170 175

Phe Cys Glu Glu Ala Arg Lys Phe Thr Phe Asn Val Ile Val Lys Gln

180 185 190

Val Leu Gly Leu Thr Pro Gln Glu Pro Glu Thr Ser Glu Ile Leu Glu

195 200 205

Asp Phe Leu Thr Phe Met Arg Gly Leu Ile Ser Leu Pro Leu Tyr Ile

210 215 220

Pro Gly Thr Pro Tyr Ala Arg Ala Val Gln Ala Arg Ser Arg Ile Ser

225 230 235 240

Ser Thr Val Lys Ala Ile Val Glu Lys Arg Arg Ala Gly Arg Arg Asn

245 250 255

Asn Asn Asn Asn Asp Asp Asp Asp Asp Asn Asn Asn Ser Lys Lys Asn

260 265 270

Ser Asp Phe Gly Gly Ile Leu Leu Ser Val Asp Thr Leu Ser Glu Asp

275 280 285

Glu Lys Val Ser Phe Val Leu Asp Ser Leu Leu Gly Gly Tyr Glu Thr

290 295 300

Thr Ser Leu Leu Met Cys Met Val Ile His Phe Leu Ser His Ser Pro

305 310 315 320

Ala Ala Leu Gln Gln Leu Lys Gln Glu His Leu Lys Ile Arg Ser Met

325 330 335

Lys Gln Lys His Asp Asp His Leu Asp Trp Glu Asp Tyr Lys Lys Met

340 345 350

Glu Phe Thr Gln Tyr Val Ile Asn Glu Ala Leu Arg Tyr Gly Asn Val

355 360 365

Val Lys Phe Val His Arg Lys Ala Leu Lys Asp Val Lys Tyr Lys Gly

370 375 380

Tyr Leu Ile Pro Ser Gly Trp Lys Val Leu Pro Val Phe Thr Ala Val

385 390 395 400

His Leu Asp Pro Ser Leu His Ala Asn Ala Thr Gln Phe His Pro Trp

405 410 415

Arg Trp Glu Ser Gln Asp Pro Thr Cys Lys Lys Phe Thr Pro Phe Gly

420 425 430

Gly Gly Ser Arg Cys Cys Pro Gly Ser Asp Leu Ala Lys Val Glu Val

435 440 445

Ala Phe Phe Leu His His Leu Val Gln Asn Phe Arg Trp Lys Thr Glu

450 455 460

Gly Glu Asp Gln Pro Ile Ala Tyr Pro Tyr Val Glu Phe Gln Arg Gly

465 470 475 480

Leu Val Leu Asn Val Asp Pro Cys Ser Glu Thr Thr Met

485 490

Claims (9)

1. A long non-coding RNA, which is long non-coding RNA as follows:

the long-chain non-coding RNA is composed of a nucleotide sequence shown in a sequence 1 in a sequence table.

2. A recombinant vector, recombinant bacterium or recombinant virus comprising the long non-coding RNA of claim 1.

3. The use of the long non-coding RNA or the coding gene regulated by the long non-coding RNA according to claim 1 for regulating the fertility of target cotton pollen;

the coding gene is any one of the following 1) -2) genes:

1) the nucleotide sequence is a DNA molecule shown from 1 st to 3334 th in a sequence 4 of a sequence table;

2) The nucleotide sequence of the DNA molecule is shown in sequence 4 in the sequence table.

4. Use of a recombinant vector, expression cassette, recombinant bacterium or recombinant virus comprising the coding gene of claim 3 for regulating fertility of target cotton pollen.

5. The recombinant vector of claim 2, wherein: the recombinant vector is obtained by inserting the long-chain non-coding RNA of claim 1 or the coding gene of claim 3 into a vector pCLCrVA;

the recombinant vector is prepared by the following method:

by usingSpeI andPac i double enzyme digestion of the vector pMD18-T containing long non-coding RNA or the coding gene, recovery of 0.5 kb target fragment, and separation of the target fragment from the vector pMD18-TSpeI andPacand (3) connecting the skeletons of the vector pCLCrVA subjected to double enzyme digestion to obtain a recombinant vector.

6. The use as claimed in claim 4 wherein the recombinant vector is a recombinant vector obtained by inserting the long non-coding DNA of claim 1 or the coding gene of claim 3 into a vector pCLCrVA;

the recombinant vector is prepared according to the method for preparing a recombinant vector as described in claim 5.

7. A method of creating a cotton male sterile line comprising the step of knocking out the long non-coding RNA of claim 1 or the coding gene of claim 3 in cotton.

8. A method for breeding transgenic cotton, which comprises knocking out the long-chain non-coding RNA of claim 1 or the coding gene of claim 3 in cotton to obtain transgenic cotton with lower pollen fertility than that of acceptor cotton.

9. The method according to claim 7 or 8, characterized in that: the knockout is achieved by the recombinant vector of claim 5 or the recombinant vector produced in claim 6.

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